Lab: DNA extraction

Deoxyribonucleic Acid, or DNA, is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, like a recipe or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments which carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

Background

DNA sculpture in Museo Principe Felipe in Valencia, Spain

Some basic, but cool, chemistry...[1]
DNA is the largest known molecule. A single unbroken strand of animal or plant DNA contains millions of atoms.[2] The DNA from a single (diploid) human cell, if the 46 chromosomes were connected end-to-end and straightened, would have a length of approximately 2 meters and a width of approximately 2.4 nanometers.[3]

Releasing the DNA...
In the DNA extraction process, the first step is to release the DNA from the cells of a living organism. Detergents and soaps are used to (1) dissolve the lipids in the cell membranes and nuclear envelope, releasing the DNA, and (2) break up proteins which may harm the DNA. (Enzymes may also be used to break up the proteins.)[2]

Clumping the DNA...
When DNA is released from a cell it typically breaks up into short strand fragments. The released DNA--highly soluble in water because the phosphate group of each nucleotide carries a negative charge--dissolves in the water. However, the positively charged sodium ions from the salt in the extraction solution are attracted to the negatively charged phosphate groups on the DNA backbone, effectively neutralizing the DNA's electric charge.

This neutralization allows the DNA molecules to aggregate with one another. When the alcohol is added, the DNA clumps together and precipitates at the water/alcohol interface because the DNA is not soluble in alcohol. The colored layer of liquid above the cell solution will contain DNA and proteins. The DNA will then move up to a layer of alcohol carefully placed above the water. DNA does not stay dissolved in alcohol, but precipitates out and will appear as a white stringy mass.[4]

Spooling the DNA on a stick...
Alcohol allows DNA fragments to stick together, or precipitate, producing a blob of DNA which you can examine. The DNA precipitate can be captured on a hook or spooled onto a wooden stick by placing the tool in the DNA and turning it. If you want to save your DNA, you can transfer it to a small container filled with alcohol.[2]

General Procedure

Print the materials and procedure below.

Try out method with wheat germ or split peas.

Design an experiment to investigate the effect of a change to the protocol (see Further experiments).

Gently, gently and even more gently, stir the mixture briefly every minute for 5 minutes. (Try NOT to create any foam.)

Remove any foam from the top of the water solution using a pipette (or eyedropper or corner of paper towel). Note that the wheat germ has settled to the bottom.

Tilt the container with the water solution at an angle. SLOWLY pour 14 ml or 1 tablespoon of alcohol down the side of the container so that it forms a layer on top of the water solution. Do not mix the two layers together. (It is crucial to pour the alcohol very slowly so that it forms a layer on top of the water solution, because DNA precipitates at the water-alcohol interface. If the alcohol mixes with the water, it will become too dilute and the DNA will not precipitate.)

Let the preparation sit for a few minutes. Watch as white, stringy, filmy DNA appears at the water and alcohol interface and floats up into the alcohol. You will usually see DNA precipitating from the solution at the water-alcohol interface as soon as you pour in the alcohol. The DNA will continue to precipitate and float upwards for about 15 min.

You may be able to get more DNA to precipitate from the solution by using one of the DNA-collecting tools (such as a glass or paper clip hook) to gently lift the mixture up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate. (An alternative procedure is to pour the water solution into a clean test tube, leaving behind the wheat germ remnants, before adding the alcohol.)

Use a wooden stick ( or a glass or paper clip hook) to collect the DNA.

Place on a paper towel.

If you want to keep the DNA, store it in 50 - 70% alcohol in a sealed tube or air dry it on a paper towel or filter paper.

Pour the blended pea mixture through a strainer into a tall container.

Add 30mL (or 2 tablespoons) of liquid detergent to the pea-liquid.

Gently swirl to mix the detergent with the pea-liquid.

Let the pea-liquid sit for 5-10 minutes.

Pour the pea-liquid into a test tube (or other small glass container) about 1/3 full.

Add a pinch of meat tenderizer (enzyme) to the test tube and stir gently (so as not to breakup the DNA strands).

Tilt the test tube and slowly pour alcohol into the tube down the side so that it forms a layer on top of the pea-liquid. Pour until you have about the same amount of alcohol in the tube as pea mixture.

Watch as DNA rises into the alcohol layer from the pea-liquid.

Use a wooden stick (or other hook) to draw the DNA into the alcohol.

Use the wooden stick to collect the DNA and place on a paper towel.

If you want to keep the DNA, store it in 50 - 70% alcohol in a sealed tube or air dry it on paper towels or filter paper.

Further experiments

The two protocols (methods) provided above differ in a few of the steps. In fact there are many different protocals which can be used to extract DNA, each of which may produce different amounts of precipitated DNA. Design an experiment using one of the ideas below[5] to test the effectiveness of variations in the protocol. Use the procedure listed at the end of this section to compare how much DNA is extracted in each experimental condition.

DNA sources

Does the food used as the DNA source effect the DNA yield? Try extracting DNA from other foods, such as kiwi, banana, liver, oatmeal, seeds, or yeast. Note that the procedure may need to be modified (e.g., longer initial soak in hot water).

Detergents

Try using different detergents and soap products to extract DNA. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?

Alcohols

Compare the amount of DNA obtained by using different alcohols (isopropyl vs. ethanol, as well as different percent concentrations).

Water temperature

Explore the effect of water temperature on DNA extraction by using different water temperatures with the protocol.

Changing the protocol

Experiment with leaving out or changing steps. When you follow the steps in an official protocol, it seems like every step is needed, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.

Do only living organisms contain DNA?

Try extracting DNA from things that you think might not have DNA.

To quantify the amount of DNA you extract:

Weigh pieces of filter paper.

Use a hook to place the DNA you extract on these pre-weighed filter paper pieces. Spread the DNA out as much as possible; it will dry more slowly if it is clumped.

Let the DNA sit for several days until you are sure it is absolutely dry.